We know of 4 glycoprotein hormones (LH, FSH, CG and TSH) and know that they interact with specific receptors that belong to the superfamily of the G protein-coupled receptors (GPCRs). The striking characteristic of the glycoprotein hormone receptors, when compared to other GPCRs, is that they contain a large leucine rich repeat-containing extracellular domain. Recently, five more GPCRs containing large leucine-repeat rich ectodomains have been cloned (LGR4-8). These GPCRs are sequentially-related to LH/FSH/TSH receptors but do not bind these hormones. They fall therefore into the class of the "orphan" GPCRs, receptors awaiting to be matched to a ligand. We hypothesize that these receptors bind thus far undescribed protein hormones and propose to isolate and characterize these hormones. We have developed a strategy that allows us to discover the natural ligands of orphan GPCRs. In short, the orphan GPCR-induced second messenger response is used to monitor the identification and purification of this orphan GPCR ligand from tissue extracts. We have preliminary data that indicate that pituitary protein extracts at various stages of purification are able to elicit a reproducible second messenger response in cells transfected with LGR4. It has also recently been discovered, by sequence analysis, that the human genome encodes two proteins (alpha2 and beta10) that are different but structurally similar to the glycoprotein hormones. We propose first to test whether alpha2 and beta10 can activate any of the orphan LGRs. We propose also to complete the isolation of the natural ligand of LGR4 and determine its sequence and isolate and sequence the natural ligands of LGR5 and 6. We will then analyze these novel ligands for their pharmacological and biological activates in vitro. Finally, we will study their roles in vivo, in particular test whether they exert a hormonal function.